Antibacterial Surfactant/Microgel Formulations, Methods of Making and Methods of Using the Same

ABSTRACT

Formulations having, as active agents, at least one quaternized polymeric microgel and at least one surfactant, methods of making, and methods of using are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application of U.S. patent applicationSer. No. 14/406,618, filed under 35 U.S.C. §371 on Dec. 9, 2014; whichis a national stage application of international application PCT/US13/44753, filed under the authority of the Patent Cooperation Treaty onJun. 7, 2013, published; which claims the benefit of U.S. PatentApplication No. 61/657,787, filed under 35 U.S.C. §111(b) on Jun. 9,2012. The entire disclosures of all the aforementioned applications areexpressly incorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under Grant No.CBET-1133795 awarded by the National Science Foundation. The UnitedStates Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Antibacterial surfactant formulations are often prepared using toxicantibacterial agents (e.g., cationic surfactants, alkyltrimethylammoniumsalts). Consequently, their use in cleaning formulations raisesenvironmental concerns.

SUMMARY OF THE INVENTION

Provided herein is an antibacterial formulation including a quaternizedpolymeric microgel comprising a cationic polymer complexed with ananionic polymer, and at least one surfactant capable of providingcolloidal stability to the formulation. In certain embodiments, thecationic polymer comprisesN-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC). Incertain embodiments, the surfactant is a nonionic surfactant. Inparticular embodiments, the nonionic surfactant comprisest-octylphenoxypolyethoxyethanol (TX-100). In certain embodiments, thesurfactant is an anionic surfactant selected from the group consistingof sodium dodecyl sulfate (SDS), sodium laurate, sodium stearate,dioctyl sodium sulfosuccinate, amphoteric sodium N-lauroyl sarcosinate,and mixtures thereof. In certain embodiments, the surfactant comprises amixture of at least one anionic surfactant and at least one nonionicsurfactant. In certain embodiments, the formulation is essentially freeof cationic surfactants. In certain embodiments, the cationic polymer ispresent at a concentration ranging from about 0.001% by weight to about10% by weight, and the anionic polymer is present at a concentration upto about 1,500 mM. In certain embodiments, the anionic polymer comprisesalginate. In certain embodiments, the anionic polymer comprisescellulose or a cellulose derivative. In certain embodiments, the anionicpolymer comprises carboxymethyl cellulose. In certain embodiments, thequaternized polymeric microgel is present at a concentration rangingfrom about 0.01% by weight to about 0.5% by weight. In certainembodiments, the quaternized polymeric microgel comprises particleshaving an average diameter ranging from about 120 nm to about 180 nm.

Further provided is a microgel mixture comprises a polyelectrolytecomplex between a quaternized chitosan polymer and an anionic polymer,where the anionic polymer is selected from the group consisting ofalginate, cellulose, and derivatives thereof. Further provided is anantibacterial formulation that includes such a microgel mixture.

Further provided is a method of making an antibacterial formulation, themethod including the steps of forming a polyelectrolyte complex betweena cationic polymer and anionic polymer, and dispersing thepolyelectrolyte complex in a surfactant to make an antibacterialformulation. In certain embodiments, the surfactant is a nonionicsurfactant. In particular embodiments, the surfactant comprises TX-100.In certain embodiments, the surfactant is an anionic surfactant selectedfrom the group consisting of sodium dodecyl sulfate (SDS), sodiumlaurate, sodium stearate, dioctyl sodium sulfosuccinate, amphotericsodium N-lauroyl sarcosinate, and mixtures thereof. In certainembodiments, the surfactant is a mixture of at least one anionicsurfactant and at least one nonionic surfactant. In certain embodiments,the cationic polymer comprises a quaternized chitosan polymer, and theanionic polymer comprises alginate. In certain embodiments, the cationicpolymer comprises a quaternized chitosan polymer, and the anionicpolymer comprises carboxymethyl cellulose. In certain embodiments, thepolyelectrolyte complex is allowed to equilibrate for a period of timebefore being dispersed in the surfactant. In certain embodiments, thesurfactant comprises TX-100 and the polyelectrolyte complex comprisesparticles of HTCC complexed with alginate. In certain embodiments, thesurfactant comprises TX-100 and the polyelectrolyte complex comprisesparticles of HTCC complexed with carboxymethyl cellulose. Furtherprovided is the product of the method for making an antibacterialformulation.

In another aspect, described herein is an antimicrobial formulationcomprised of: at least one nonionic surfactant capable of providingcolloidal stability to the formulation, and at least one quaternizedpolymeric microgel capable of being dispersed in the nonionicsurfactant. The quaternized polymeric microgel is comprised of one ormore of: chitosan oligomers or chitosan polymers. Further, theantimicrobial formulation is essentially free of cationic surfactants.

In certain embodiments, the chitosan oligomer or chitosan polymercomprises one or more of: chitin, a chitin derivative, chitosan,chitosan derivatives, and cross-linked quaternized chitosans orderivatives thereof.

In certain embodiments, the formulation consists essentially of thenonionic surfactant and the quaternized polymeric microgel, wherein theconcentration of the quaternized polymeric microgel ranges from about0.01 wt. % to about 0.5 wt. %., per volume in water.

In certain embodiments, the formulation consists essentially of thenonionic surfactant and the quaternized polymeric microgel, wherein theconcentration of the quaternized polymeric microgel is about 0.1 wt. %,per volume in water.

In certain embodiments, the nonionic surfactant is present at aconcentration, based on the weight of the formulation, of: about 250mmol/kg or less; about 100 mmol/kg or less; about 25 mmol/kg or less;about 15 mmol/kg or less; or, about 5 mmol/kg or less.

In certain embodiments, the nonionic surfactant comprises one or moreof: polyoxypropylene glycol alkyl ethers, and glycol octylphenol ethers.In certain embodiments, the nonionic surfactant comprises one or moreof: ethoxylated alkylphenols, glucoside alkyl ethers, andpolyoxyethylene glycol alkylphenol ethers. In certain embodiments, theethoxylated alkyphenols comprise ethoxylated octylphenol.

In certain embodiments, the nonionic surfactant comprisest-octylphenoxypolyethoxyethanol (TX-100), and wherein the quaternizedpolymeric microgel comprisesN-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC).

In certain embodiments, the formulation comprises:t-octylphenoxypolyethoxyethanol (TX100) at a concentration ranging fromabout 0.05% to about 1%, by weight, per volume in water; and,N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC) at aconcentration ranging from about 0.3% to about 3%, by weight, per volumein water.

In certain embodiments, the quaternized polymeric microgel is preparedthrough ionic cross-linking of quaternized chitosan with sodiumtripolyphosphate (TPP).

In certain embodiments, the quaternized polymeric microgel comprisesparticles having a size distribution of about 10 nm to about 1000 nm;about 200 nm to about 400 nm; or, about 10 nm to about 100 nm.

In certain embodiments, the antimicrobial formulation comprises: atleast one nonionic surfactant present at a total concentration in theantimicrobial formulation in the range of from about 0.05% to about 3%,by weight, per volume in water; and, one or more microgels comprisingquaternized polymeric microgel comprised of one or more of: chitosanoligomers or chitosan polymers; the total concentration of thequaternized polymeric microgels in the antimicrobial formulation rangingfrom about 0.01% to about 0.15%, by weight, per volume in water.

In certain embodiments, the nonionic surfactant is present in admixturewith at least one anionic surfactant, wherein the anionic surfactant ispresent at a concentration wherein amine groups on the quaternizedpolymeric microgel are not neutralized.

In certain embodiments, the anionic:nonionic ratio is about 30:70. Incertain other embodiments, the anionic:nonionic ratio is about 40:60.Also, in certain embodiments, the anionic surfactant is present at aconcentration, based on the weight of the formulation of: about 2mmol/kg or less; about 1 mmol/kg or less; or, about 0.5 mmol/kg or less.

In certain embodiments, the surfactant admixture comprises about 40:60anionic:nonionic surfactant, and wherein the microgel is dispersed inabout 3 mmol/kg or less, based on the weight of the formulation, of thesurfactant admixture.

In certain embodiments, the anionic surfactant comprises one or morealkali metal C₈₋₁₈ alkyl sulfates. In certain embodiments, wherein thealkali metal C₈₋₁₈ alkyl sulfates are selected from the group consistingof: sodium lauryl sulphate, sodium laureth sulfates, alkyl ethersulfates, alkyl benzene sulfonates, fatty acids, and fatty acid salts.

In another aspect, described herein are uses of the formulation wheresuch formulation is in the form of a tablet, powder, gel, capsule,liquid, coating, film, foam, sponge, woven material, non-woven material,textile material, knitted material, porous material or solid material.

In another aspect, described herein are articles comprising a porous orsolid material at least partially coated with the formulation; and/or atleast partially comprised of the formulation.

Other systems, methods, features, and advantages of the presentinvention will be or will become apparent to one with skill in the artupon examination of the following drawings and detailed description. Itis intended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe present invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIG. 1: ITC data for (♦) sodium dodecyl sulfate (SDS) and (▪)t-octylphenoxy polyethoxyethanol (TX100) binding to chitosan-basedmicrogels. (The lines are guides).

FIG. 2: Microgel ζ-potentials in the presence of (♦) SDS and (▪) TX-100.(The lines are guides).

FIGS. 3A-3B: Dynamic light scattering analysis of cationic microgeldispersions in SDS:TX100 solutions with (♦) 100:0 (▪) 70:30. (

) 40:60 and (

) 10:90 SDS:TX100 molar ratios, shown as function of (FIG. 3A) SDSconcentration and (FIG. 3B) total surfactant concentration (with therapid precipitation transition indicated by the shaded region). Thesolid lines are guides.

FIG. 4: DLS analysis of microgel size distributions in: (a) 0 mmol/kg,(b) 2.1 mmol/kg, (c) 4.2 mmol/kg, (d) 6.1 mmol/kg, (e) 8.0 mmol/kg, (f)9.8 mmol/kg, (g) 11.5 mmol/kg, and (h) 14.4 mmol/kg TX100 solutions. Thecurves were offset vertically for clarity.

FIG. 5: Normalized Pseudomonas aeruginosa viable cell counts after 0-30min of exposure to microgels mixed with: (1) water, (2) 1.0 mmol/kg SDS,(3) 3.0 mmol/kg 40:60 SDS:TX100 mixture, (4) 10 mmol/kg SDS, and (5)14.4 mmol/kg TX-100, and microgels-free solutions containing (6) 10mmol/kg SDS, (7) 14.4 mmol/kg TX-100, (8) 3.0 mmol/kg 40:60 SDS:TX100mixture, and (9) water.

FIGS. 6A-6B: Visual observation (FIG. 6A) and spectroscopicquantification (FIG. 6B) of guaiazulene dye solubilization insurfactant/microgel dispersions containing (i) 1.75 mmol/kg SDS, (ii)1.12 mmol/kg 70:30 SDS:TX-100, (iii) 1.04 mmol/kg 40:60 SDS:TX-100, (iv)1.09 mmol/kg 10:90 SDS:TX-100, and (v) 14.4 mmol/kg TX-100. Thespectroscopic analysis was performed after filtering out the microgelsand larger dispersed dye particles from the samples.

FIGS. 7A-7B: Photographs (FIG. 7A) and turbidimetry measurements (FIG.7B) showing the effects of surfactant at (i) 100:0, (ii) 70:30, (iii)40:60, (iv) 10:90 and (v) 0:100 SDS:TX100 ratios on the clarity ofsurfactant/microgel dispersions.

FIG. 8: Table 1. Concentrations of SDS and total surfactant at the onsetof precipitation.

FIG. 9: DLS data showing the PDI-values of cationic microgel dispersionsin SDS/TX-100 solutions with: (♦) 100:0 (▪) 70:30, (

) 40:60 and (

) 10:90 SDS:TX100 molar ratios, as function of SDS concentration. Thesolid lines are guides to the eye.

FIG. 10: The ζ-potentials of cationic microgel dispersions in SDS/TX100solutions with (♦) 100:0 (▪) 70:30, (

) 40:60 and (

) 10:90 SDS:TX100 molar ratios, plotted as function of SDSconcentration.

FIG. 11: ITC data for the titration of 40 mmol/kg SDS into (▪) 0.082 wt% molecular HTCC and (

) HTCC/TPP microgels (also containing 0.082 wt % HTCC) mixtures. Thelines are guides to the eye, while the shaded region indicates thesaturation transition.

FIG. 12: ITC data for the titration of 0.4 wt % TPP into (▪) 0.1 wt %HTCC and (

) 0.1 wt % chitosan solutions. The lines are guides to the eye.

FIG. 13: A photograph of guaiazulene blue dye mixed with 1.75 mmol/kgSDS solution.

FIGS. 14A-14C: Graphs showing the size distributions (z-averagediameters (FIG. 14A) and polydispersity indices (PDI, FIG. 14B)) andzeta potentials (FIG. 14C) of the HTCC/alginate particles prepared.

FIGS. 15A-15D: Graphs showing the z-average diameters (FIG. 15A),polydispersity indices (FIG. 15B), derived count rate (DCR, FIG. 15C),and zeta potential (FIG. 15D), each as a function of monomer:alginateratio, at varying concentrations of HTCC in the microgel mixtures.

FIGS. 16A-16B: Graphs showing the antibacterial activity ofHTCC/alginate formulations. FIG. 16A shows the percent reduction inviable bacterial cells versus time. FIG. 16B shows the log reduction ofviable bacterial cells versus time, with cases having 100% reductionbeing assigned the arbitrary log reduction value of 6.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

The use of the word “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value. In any embodiment discussed inthe context of a numerical value used in conjunction with the term“about,” it is specifically contemplated that the term “about” can beomitted.

The terms “a” and “an,” when used in conjunction with the word“comprising” in the claims or specification, denotes one or more, unlessspecifically noted.

All percentages, parts and ratios are based upon the total weight of thecompositions of the present invention, unless otherwise specified. Allsuch weights as they pertain to listed ingredients are based on theactive level and, therefore, may not include solvents or by-productsthat may be included in commercially available materials, unlessotherwise specified. The term “weight percent” may be denoted as “wt. %”herein. All molecular weights as used herein are weight averagemolecular weights, unless otherwise specified.

The terms “nil-cationic surfactant” or “substantially or essentiallyfree of a cationic surfactant,” as used herein, means that theformulations comprise very low levels of cationic surfactant, andpreferably no cationic surfactant. For example, “essentially free” canmeans less than 1% by weight, preferably less than 0.5% by weight, morepreferably less than 0.1% by weight, and even more preferably less than0.01% by weight of a component based on the total weight of thecomposition.

The term “antimicrobial” generally refers to the microbicidal ormicrobistatic properties of a formulation that enables such to kill,destroy, inactivate, or neutralize a microorganism; or to prevent orreduce the growth, ability to survive, or propagation of amicroorganism.

The term “zeta-potential” or “ζ-potential” generally refers to theelectrical potential that exists across the interface of all solids andliquids, e.g., the potential across the diffuse layer of ionssurrounding a charged colloidal particle. Zeta potential can becalculated from electrophoretic mobilities, i.e., the rates at whichcolloidal particles travel between charged electrodes placed in contactwith the substance to be measured, using techniques well known in theart.

The term “surfactant” as used herein includes dispersing agents,suspending agents, emulsion stabilizers and detergents.

Nonionic Surfactants

In particular embodiments, “surfactant” includes non-ionic surfactants.Non-limiting examples of nonionic surfactants include: polyoxypropyleneglycol alkyl ethers, and glycol octylphenol ethers; ethoxylatedalkylphenols, glucoside alkyl ethers, and polyoxyethylene glycolalkylphenol ethers, such as ethoxylated octylphenol.

In one embodiment, the nonionic surfactant comprisest-octylphenoxypolyethoxyethanol (other names4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol,t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenylether) (C₁₄H₂₂O(C₂H₄O)_(n)) (also known as Triton X-100 or TC-100) is anonionic surfactant that has a hydrophilic polyethylene oxide chain (onaverage it has 9.5 ethylene oxide units) and an aromatic hydrocarbonlipophilic or hydrophobic group. The hydrocarbon group is a4-(1,1,3,3-tetramethylbutyl)-phenyl group.

In certain embodiments, the formulations comprise up to about 250mmol/kg of nonionic surfactant. In particular embodiments, theformulations comprise a nonionic surfactant present at a concentrationof about 100 mmol/kg, or about 25 mmol/kg, or about 15 mmol/kg, or about5 mmol/kg.

In certain embodiments, the formulations comprise nonionic surfactant inconcentrations ranging from about 0.05% w/v in water to about 3% w/v inwater.

In certain other embodiments, the formulations comprise nonionicsurfactant in concentrations ranging from about 0.05% w/v in water toabout 50% w/v in water.

Microgels

The term “quaternized polymers” generally refers to the building blocksfor the microgels. In certain embodiments, the quaternized polymerscomprise quaternized cellulose derivatives. In certain otherembodiments, the quaternized polymers comprise chitosan and derivativesthereof. In certain other embodiments, bio-polymers other than chitosanare also possible, such as agarose and dextran.

The term “microgels” includes the quaternized polymers, as describedherein. Also, in certain embodiments, the microgels can be comprised ofsynthetic polymers with quaternary amine sidechain groups. In stillother embodiments, the microgels can be prepared though the covalent orionic cross-linking of synthetic polymers with cationic monomer units,through polymerization of multifunctional monomers, or throughpolyelectrolyte complexation. For example, while not wishing to be boundby theory, it is believed that chitosan has antibacterial activity dueto the amine groups on its glucosamine monomer units, and is furtherstrengthened through the substitution of the glucosamine primary amineswith quaternary amines. In addition to enhancing the antibacterialactivity, this “quaternization” of chitosan also enhances its solubilityat neutral and alkaline pH.

In a particular embodiment, chitosan (or its quaternized derivatives,e.g., trimethylchitosan, or quaternized chitosan) is prepared ascross-linked particles (i.e., micro- or nanogels) which increases theantibacterial activity greater than that of the chitosan derivatives inthe molecular form. In other embodiments, chitosan is prepared aspolyelectrolyte-complexed particles with an oppositely charged polymer,such as alginate or carboxymethyl cellulose.

The term “chitosan” as used herein generally includes a cationicpolyelectrolyte derived from the naturally abundant biopolymer, chitin.In certain embodiments, chitosan is composed of cationic D-glucosamineand nonionic N-acetyl-D-glucosamine monomer units, and is soluble inacidic aqueous solutions (at pH<6), where the pH-sensitive primary aminegroups on the glucosamine monomer units become charged. This switchableamine group enables chitosan to form a variety of gel-like structuresthrough its exposure to alkaline solutions, or its complexation withoppositely-charged counterions, polymers, and surfactants.

Suitable chitosan derivatives include, but are not limited to:N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC);hydroxyalkyl chitosans, such as hydroxypropyl chitosan; N,N,N-trimethylchitosan (TMC); N-propyl-(QuatPropyl), N-butyl-(QuatButyl) and N-hexyl(QuatHexyl)-N,N-dimethyl chitosan;N-(4-N,N-dimethylaminocinnamyl)chitosan chloride; N,N-octylchitosan;N-benzylchitosan; N-(4-methylbenzylchitosan);N-(4-hydroxybenzyl)chitosan; N-(2-methoxybenzyl)chitosan;N-(4-methoxybenzyl)chitosan; N-(3,4-dimethoxybenzyl)chitosan;N-(4-fluorobenzyl)chitosan; N-(4-chlorobenzyl)chitosan;N-(3-bromobenzyl)chitosan; N-(4-bromobenzyl)chitosan;N-(4-trifluorobenzyl)chitosan; N-(4-nitrobenzyl)chitosan;N-(4-carboxybenzyl)chitosan; N-(4-pyridinylmethyl)chitosan;N-(2-thiophenylmethyl)chitosan, and combinations thereof.

In certain embodiments, the quaternized polymeric microgel comprises asaccharide. In certain embodiments, the quaternized polymeric microgelcomprises a biodegradable linear polysaccharide having glucosamineunits. In certain embodiments, the quaternized polymeric microgel isselected from chitosan, chitosan derivatives, cellulose, chitin, chitinderivatives, and combinations thereof.

The quaternized chitosan microgels are useful in the preparation ofantibacterial surfactant formulations for use in cleaning and personalcare products. For example, dilute chitosan-based microgel formulationsare useful as replacements or substitutes for cationic surfactants andother toxic small molecule antibacterial agents (e.g., triclosan orbleach) in personal care and/or cleaning products. The quaternizedchitosan microgels are antibacterial even at very low concentrations andare cytocompatible with mammalian cells. In the formulations describedherein, the antibacterial cationic microgels are dispersed in non-toxicnonionic surfactant solutions. In certain embodiments, theseformulations achieve desirable antibacterial activity without the use ofcationic surfactants or other small molecule antibacterial agents, whichcationic surfactants are often toxic.

The formulations further comprise a quaternized polymeric microgel at aconcentration ranging from about 0.01% by weight to about 0.5% byweight, or more preferably from about 0.1% by weight to about 0.3% byweight.

In certain embodiments, the formulations comprise a quaternizedpolymeric microgel at concentrations ranging from about 0.01% w/v inwater to about 0.15% w/v in water.

In other certain embodiments, the formulations comprise a quaternizedpolymeric microgel at concentrations ranging from about 0.01% w/v inwater to about 20% w/v in water, and in other embodiments, to about 50%.It is to be understood, that in certain embodiments, the volume mayrange from about, at least about, or at most about 0.1%, 0.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%,9%, 9.5%, 10%, and up to about 20%, or up to about 50%, or any rangederivable therein.

In certain embodiments, the quaternized polymeric microgel has azeta-potential of about 15 mV or less. In certain embodiments, thequaternized polymeric microgel has a size distribution of about 10 nm toabout 1000 nm, or from about 200 nm to about 400 nm, or from about 10 nmto about 100 nm.

Antimicrobial Formulations

Also described herein are formulations having quaternized chitosanmicrogels where the active agents (comprising the microgels and anonionic surfactant) are present at concentrations effective to reducebacterial counts.

The surfactant solubilizes hydrophobic compounds (e.g., oil in emulsionsor different types of soils on clothing), while the microgels providestrong antibacterial activity without the use of cationic surfactants orother types of toxic small molecule antibacterial agents (such astriclosan or bleach). The nonionic surfactant (1) does not undermine theantibacterial activity of the microgels; and (2) does not cause themicrogels to precipitate out of the mixture.

In certain embodiments, the formulations exhibit high antimicrobialactivity, thus allowing for the use of low concentrations of the activeagents to obtain rapid killing of large numbers of microbes uponcontact. Accordingly, the low concentration formulations disclosedherein have surprising activity in reducing microbial populations on thesurface of an article without impacting the properties of the article'ssurface.

Polyelectrolyte Complexation

In some particular embodiments, instead of using small ions as ioniccrosslinkers or covalent crosslinking agents, biocompatible microgelsfor the antibacterial surfactant formulations are prepared throughpolyelectrolyte complexation by complexing an antimicrobial polycationwith an oppositely charged polymer. In one non-limiting example, theantibacterial cationic polymer is complexed with a non-toxic anionicpolymer such as alginate or carboxymethyl cellulose. Thispolyelectrolyte complexation approach for preparing antibacterialsurfactant/microgel formulations presents many advantages. First, it isa cleaner procedure for preparing antibacterial surfactant/microgelmixtures. Polyelectrolyte complexation uses ecofriendly ingredients toprepare the microgels, as opposed to phosphate derivatives (which cancontribute to the eutrophication of lakes), organic solvents, or toxiccrosslinking agents. The use of phosphate derivatives, organic solvents,or toxic crosslinking agents results in a more costly andless-ecofriendly preparation process than polyelectrolyte complexation.Second, polyelectrolyte complexation is a simple and inexpensiveprocess. Third, the multivalent interactions between the anionic andcationic polymers impart the microgels with greater stability comparedto ionically crosslinked microgels. This improved stability is conduciveto the microgels being formulated in anionic and anionic/nonionicsurfactant mixtures, as opposed to only nonionic surfactants. Fourth,the polyelectrolyte complexation method can be conducted without the useof cationic surfactants, such as alkyltrimethylammonium salts, thattypical antibacterial surfactant formulations use. Because suchsurfactants are toxic, their use in cleaning formulations raisesenvironmental concerns.

A non-limiting example of a procedure for preparing asurfactant/microgel formulation with microgel prepared throughpolyelectrolyte complexation involves adding a solution of a non-toxicanionic polymer, such as alginate, to a solution of a quaternizedchitosan polymer. This mixture is allowed to equilibrate to form amicrogel mixture. After equilibration, a surfactant such as TX100 isadded to the microgel mixture. In certain embodiments, the microgelmixture exhibits a positive zeta potential of about 30 mV before thesurfactant is added.

Suitable non-toxic anionic polymers for use in a polyelectrolytecomplexation method include, but are not limited to: alginates,celluloses, hyaluronan, dextran sulfate, poly(acrylic acid),poly(methacrylic acid), poly(styrene sulfate), poly(vinyl sulfonate),heparin, cellulose phthalate acetate, chondroitin sulfate, and the like.The term “alginate” is used as the general name given to alginic acidand its salts. Alginates are composed of D-mannosyluronic(mannuronic-“M”) and L-gulopyranosyluronic (guluronic-“G”) acidresidues. Modified alginates, such as esterified alginates, are alsoincluded. Celluloses are linear chain polysaccharides of D-glucoseunits. Modified celluloses, such as carboxyalkyl celluloses, includingcarboxymethyl cellulose, are particularly suitable for use in themicrogel formulations described herein.

The quaternized chitosan polymer can be composed of any suitablechitosan derivative, as described above. Suitable chitosan derivativesinclude, but are not limited to:N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC);hydroxyalkyl chitosans, such as hydroxypropyl chitosan; N,N,N-trimethylchitosan (TMC); N-propyl-(QuatPropyl), N-butyl-(QuatButyl) andN-hexyl(QuatHexyl)-N,N-dimethyl chitosan;N-(4-N,N-dimethylaminocinnamyl)chitosan chloride; N,N-octylchitosan;N-benzylchitosan; N-(4-methylbenzylchitosan);N-(4-hydroxybenzyl)chitosan; N-(2-methoxybenzyl)chitosan;N-(4-methoxybenzyl)chitosan; N-(3,4-dimethoxybenzyl)chitosan;N-(4-fluorobenzyl)chitosan; N-(4-chlorobenzyl)chitosan;N-(3-bromobenzyl)chitosan; N-(4-bromobenzyl)chitosan;N-(4-trifluorobenzyl)chitosan; N-(4-nitrobenzyl)chitosan;N-(4-carboxybenzyl)chitosan; N-(4-pyridinylmethyl)chitosan;N-(2-thiophenylmethyl)chitosan, and combinations thereof.

Any of the surfactants described above can be used to prepare asurfactant/microgel formulation with the microgels prepared bypolyelectrolyte complexation. TX100 is particularly useful for thispurpose, but any suitable polyoxypropylene glycol alkyl ether, glycoloctylphenol ether, ethoxylated alkylphenol, glucoside alkyl ether,polyoxyethylene glycol alkylphenol ether (such as ethoxylatedoctylphenol), or mixture thereof can be used as the surfactant. Inaddition, the microgels prepared by polyelectrolyte complexation haveadded stability that allows them to be mixed with anionic oranionic/nonionic surfactant mixtures. This greatly expands the range ofapplications where the microgels can be used. Suitable anionicsurfactants for use in a formulation with the microgels alone or in amixture with one or more nonionic surfactants include, but are notlimited to: sodium dodecyl sulfate (SDS), sodium laurate, sodiumstearate, dioctyl sodium sulfosuccinate, amphoteric sodium N-lauroylsarcosinate, and mixtures thereof.

In certain embodiments, the formulations contain surfactant at aconcentration up to about 1,500 mM. In certain embodiments, theformulations contain surfactant at a concentration up to about 500 mM.In certain embodiments, the formulations comprise up to about 250mmol/kg of nonionic surfactant. In particular embodiments, theformulations comprise a surfactant present at a concentration of about100 mmol/kg, or about 25 mmol/kg, or about 15 mmol/kg, or about 5mmol/kg. In certain embodiments, the formulations comprise surfactant inconcentrations ranging from about 0.05% w/v in water to about 3% w/v inwater. In certain other embodiments, the formulations comprisesurfactant in concentrations ranging from about 0.05% w/v in water toabout 50% w/v in water.

The formulations containing microgels prepared by polyelectrolytecomplexation generally include a quaternized polymeric microgel at aconcentration ranging from about 0.001% to about 10% by weight (forexample, in the form of a concentrate), or from about 0.01% by weight toabout 0.5% by weight, or more preferably from about 0.1% by weight toabout 0.3% by weight. In certain embodiments, the formulations comprisea quaternized polymeric microgel at concentrations ranging from about0.01% w/v in water to about 0.15% w/v in water. In other certainembodiments, the formulations comprise a quaternized polymeric microgelat concentrations ranging from about 0.01% w/v in water to about 20% w/vin water, and in other embodiments, to about 50%. It is to beunderstood, that in certain embodiments, the volume may range fromabout, at least about, or at most about 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%,3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%,10%, and up to about 20%, or up to about 50%, or any range derivabletherein.

In certain embodiments, the quaternized polymeric microgel has azeta-potential of about 15 mV or less. In certain embodiments, thequaternized polymeric microgel has a size distribution of about 10 nm toabout 1000 nm, or from about 200 nm to about 400 nm, or from about 10 nmto about 100 nm.

The surfactant/microgel formulations prepared with microgels formed frompolyelectrolyte complexation are useful for the same purposes as thesurfactant/microgel formulations prepared with microgels formed fromionic or covalent crosslinking. This is because the microgels haveantibacterial properties whether prepared by polyelectrolytecomplexation or by ionic or covalent crosslinking Thus, the quaternizedchitosan microgels prepared from polyelectrolyte complexation are usefulin the preparation of antibacterial surfactant formulations for use incleaning and personal care products. For example, dilutepolyelectrolyte-complexed chitosan-based microgel formulations areuseful as replacements or substitutes for cationic surfactants and othertoxic small molecule antibacterial agents (e.g., triclosan or bleach) inpersonal care and/or cleaning products. The polyelectrolyte-complexedquaternized chitosan microgels are antibacterial even at very lowconcentrations and are cytocompatible with mammalian cells.

In certain embodiments of the formulations described herein, theantibacterial cationic microgels prepared from polyelectrolytecomplexation are dispersed in non-toxic nonionic surfactant solutions.These formulations achieve desirable antibacterial activity without theuse of cationic surfactants or other small molecule antibacterialagents, which cationic surfactants are often toxic.

Concentrates

Any of the formulations described herein, including those havingmicrogels prepared by polyelectrolyte complexation, can be prepared asconcentrates. By way of non-limiting example, the formulations could beprepared with concentrations of nonionic surfactant up to about 2,500mmol/kg, and with quaternized polymeric microgels at concentrations upto about 10% by weight and concentrations of surfactant up to about1,500 mM. In some non-limiting examples, the concentrates may have anoverall surfactant/microgel content of 60% by weight or more. Theconcentrates can then be diluted down to the desired concentrations ofactive agents prior to use.

In one non-limiting example, a surfactant/microgel formulation may bepackaged in a concentrate containing up to about 10% by weight ofquaternized chitosan particles prepared by polyelectrolyte complexation.The concentrate can then be diluted to sub-100 ppm concentrations. Thereare many other methods of preparing the formulations described hereinthat will be readily apparent to the skilled practitioner.

Surfactants Comprised of Anionic/Nonionic Admixtures

In certain embodiments, the nonionic surfactant is present in admixturewith at least one anionic surfactant. The ratio of anionic:nonionicsurfactant can be about 0:100. In particular embodiments, theanionic:nonionic surfactant ratio is 10:90, 20:80, 30:70, or 40:60. Incertain embodiments, the concentration of the anionic surfactant is 2mmol/kg or less, 1 mmol/kg or less, or 0.5 mmol/kg or less. Non-limitingexamples of anionic surfactants include, for example, sodium dodecylsulfate (SDS), sodium laureth sulfate (SLS), disulfosuccinate (DSS),alkyl benzene sulphonates, sulphated fatty alcohols, fatty acids andfatty acid salts, and the like.

Methods

Further described herein are methods of formulating chitosan-basedmicrogel formulations with surfactants. Such methods overcome the issuesthat arise due to the electrostatic binding between oppositely chargedsurfactants and polymers. In addition, this binding can lead to theprecipitation of the surfactant/polymer complex, thereby complicatingthe formulation of single-phase surfactant/microgel formulations.Furthermore, by binding to the chitosan, the surfactant may reduce theavailability of the cationic amine groups that underlie the biocidalactivity of the microgels (which could inactivate their antibacterialproperties).

Also described herein are antibacterial surfactant/microgel formulationsthat use one or more of anionic, nonionic, and anionic/nonionicsurfactant systems. The binding of anionic and nonionic surfactants tothe microgels was probed by electrophoretic light scattering andisothermal titration calorimetry (ITC). The surfactant/chitosan-basedmicrogel formulations were determined to have desirable: (1) colloidalstability, which is essential for maintaining the formulation in asingle phase; (2) antibacterial activity, which is necessary toinactivate bacteria using minimal chitosan quantities; (3) hydrophobesolubilization properties, which reflect the ability to remove soils ordeliver essential oils; and (4) the effect of surfactants on dispersionclarity, which is often desirable for designing aesthetically-appealingproducts.

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. All publications, including patentsand non-patent literature, referred to in this specification areexpressly incorporated by reference. The following examples are intendedto illustrate certain preferred embodiments of the invention and shouldnot be interpreted to limit the scope of the invention as defined in theclaims, unless so specified.

EXAMPLES

Materials and Methods

Materials

All experiments were performed using Millipore Direct Q-3 deionizedwater (18.0-18.2 MQ.m resistivity). Chitosan (90% degree ofdeacetylation, as determined by pH titration), sodium tripolyphosphate(TPP), t-octylphenoxypolyethoxyethanol (Triton X-100®) (TX100),glycidyltrimethylammonium chloride, and guaiazulene were purchased fromSigma-Aldrich (St. Louis, Mo.). Ultrapure sodium dodecyl sulfate (SDS)was purchased from MP Biomedicals (Solon, Ohio), and sodium chloride(NaCl) was purchased from Fisher Scientific (Fair Lawn, N.J., USA). Allmaterials were used as received.

Synthesis of Quaternized Chitosan

The antibacterial quaternized chitosan derivative,N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC), wasprepared. Briefly, 2.0 g of chitosan flakes were dispersed in 38 mL ofwater at 85° C. and agitated with a magnetic stirrer. Three 2.45 mLaliquots of glycidyltrimetylammonium chloride were then added to thedispersion at 2 h intervals, and allowed to react for 12 h. Thequaternization then became apparent from the dissolution of chitosanflakes, which are otherwise insoluble at neutral and high pH. After thereaction, the HTCC was purified by dialyzing once against 10 mM NaCl andthrice against deionized water (for 12 h each time) through aSpectralPor regenerated cellulose membrane (molecular weightcutoff=2,000). The final product was then freeze dried for 48 h on aLabconco Freeze Dryer 3 lyophilizer (Kansas City, Mo.) and characterizedby ¹H NMR to confirm that the product spectra (not shown) matched theliterature spectra for HTCC.

Microgel Preparation

The quaternized chitosan microgels were prepared through the ioniccross-linking of HTCC with TPP. Briefly, 3.3 mL of 0.1 wt % TPP solutionwere added dropwise (at a rate of 200 RL/min) to 15 mL of 0.1 wt % HTCCsolution, where each solution contained 10 mM NaCl. The addition ratewas controlled with a Fisher Scientific syringe pump (Model #78-01001),and the receiving HTCC solution was stirred with a cylindrical (12 mm×4mm) magnetic stir bar at 800 RPM inside a 20 mL scintillation vial. Themicrogels were then allowed to equilibrate for 15 min, whereupon theirsize distributions and ζ-potentials were characterized by dynamic andelectrophoretic light scattering.

Isothermal Titration Calorimetry

The binding of the anionic surfactant SDS and the non-ionic surfactantTX100 to the microgels was tested by isothermal titration calorimetry(ITC), using a Microcal VP-ITC instrument (GE Healthcare, Northampton,Mass.). In each measurement, twenty five 10-μL injections of 40 mmol/kgsurfactant (either SDS or TX100) solution (containing 10 mM NaCl) wereadded to a 1.48 mL sample cell filled with microgel dispersions at amatching NaCl concentration. To account for the heat of surfactantdilution (and demicellization), control measurements were alsoperformed, where 40 mM surfactant solutions were injected intomicrogel-free 10 mM NaCl solutions. The enthalpic signal due to bindingwas then obtained (as the heat absorbed from the sample cell per mole ofadded surfactant) by subtracting this heat of dilution from the signalobtained from the addition of surfactant to the microgels.

Dynamic and Electrophoretic Light Scattering

To determine the effect of surfactant on the size distribution andsurface charge of the chitosan-based microgels, the dispersions wereprobed by dynamic and electrophoretic light scattering. Thesemeasurements were performed on a Zetasizer Nano ZS (Malvern,Worcestershire, UK). The effect of surfactant binding on the surfacecharge was inferred from the changes in potential, which were estimatedfrom the electrophoretic light scattering measurements using theHelmholtz-Smoluchowski equation. The effects of SDS and SDS:TX-100surfactant systems on size distributions (which reflected the microgelaggregation states) was measured by dynamic light scattering (DLS),based on changes in the z-average hydrodynamic diameters (estimated viacumulant analysis). Conversely, the effects of TX100 on microgelaggregation were characterized by analyzing the DLS data by the multiplenarrow modes algorithm. This was because the microgels remaineddispersed in a single phase even at high surfactant concentrations(where the scattering from the micelles became significant), and themultiple narrow modes algorithm allowed the scattering signal from themicrogels to be resolved from that of the TX100 micelles.

Colloidal Stability of Surfactant/Microgel Formulations

The short-term colloidal stability of surfactant/microgel formulationswas characterized through visual observation and DLS. Concentratedsurfactant and surfactant admixtures (containing either 10 mmol/kg SDS,14.3 mmol/kg 70:30 SDS:TX-100; 25 mmol/kg 40:60 SDS:TX-100, 100 mmol/kg10:90 SDS:TX-100, or 100 mmol/kg TX100) were titrated into the microgeldispersions in 65- to 250 μL increments. During the titrations thesurfactant/microgel formulations were continuously stirred at 800 RPMusing a cylindrical (12 mm×4 mm) magnetic stir bar, and equilibrated for6 min after each surfactant addition prior to visual analysis and DLScharacterization. Each titration was performed three times and yieldedreproducible results. The long-term colloidal stability was then testedby equilibrating the samples at room temperature over 6 weeks andmonitoring them for precipitation.

Antibacterial Activity Tests

The effect of anionic and nonionic surfactants on the microgelantibacterial properties was probed using Pseudomonas aeruginosa(wild-type P. aeruginosa PAOI strain) as model Gram-negative bacteria.The bacteria were cultured in one-tenth strength LB broth (2.5 g/L,Difco Laboratory, Detroit, Mich.) at 37° C. until the late-exponentialphase. Bacterial cells were then harvested by centrifugation at 2,000 gfor 15 min (using an Eppendorf 5804R centrifuge; Hamburg, Germany),washed and resuspended in phosphate buffer (0.54 g Na₂HPQ₄ and 0.88 gKH₂PO₄ per liter, pH=6.98) as bacterial suspensions (0D₆₀₀=0.25±0.02).

All glassware used in this example was sterilized in an autoclave.Likewise, the HTCC, TPP, and surfactant solutions used to prepare thesurfactant/microgel formulations were sterile-filtered using SantoriusMinisart NML 0.8 μm syringe filters. The microgels were filtered, andafter the filtration, the microgels were prepared inside a BSL 2certified biosafety chamber, whereupon 2 mL of surfactant (whichcontained either SDS, TX-100, or an admixture of both) were added at arate of 1 mL/min, such that the final HTCC concentration was 7.4×10⁻² wt%. Each antimicrobial formulation sample was then tested in triplicateat room temperature (22±2° C.). The bacterial suspension was mixed withthe surfactant/microgel formulations in a 1:1000 ratio (e.g., 15 μl:15ml). After mixing, samples were taken at 5, 15, and 30 min to quantifythe effect of surfactant/microgel formulations on the number of viablecells. The viable cells were enumerated at each time point using theheterotrophic plate count method with R2A agar plates (DifcoLaboratories, Detroit, Mich.).

Dye Solubilization Tests

To test the hydrophobe solubilization properties of eachsurfactant/microgel formulation type, surfactant/microgels formulationswere prepared as described previously using SDS:TX100 ratios of 100:0,70:30, 40:60, 10:90, and 0:100, and total surfactant concentrations of1.75, 1.12, 1.04, 1.08 and 14.4 mmol/kg, respectively. Theconcentrations of SDS-containing samples corresponded to the maximumsurfactant concentrations that were mixed with the microgels during thetitrations before phase separation occurred. Conversely, theconcentration of 14.4 mmol/kg was used for the 0:100 SDS:TX100 system(in which phase separation did not occur) because it was the highestsurfactant concentration tested in the colloidal stability and biocidalactivity experiments. Three milliliters of each formulation type wereadded to a test tube and mixed with 0.2 g of hydrophobic guauazulene dyefor 5 minutes on a vortex mixer. The samples were then equilibrated for24 hours and—after separating the microgels and undissolved dye particleby passing the formulation though a Millipore Millex® FG 0.2 μm filter(to minimize artifacts due to light scattering and absorption from themicrogels and undissolved dye)—quantified for the dissolved dye contentby UV/Vis spectroscopy (extinction coefficient=0.2575 mM⁻¹ cm⁻¹) using aCary 50 spectrophotometer. Each measurement was performed in triplicate.

Turbidimetric Measurements

To measure the effect of surfactant choice on formulation clarity, theturbidity of each formulation used in the dye solubilization experimentwas quantified. This choice of formulation compositions, whichcorresponded to the maximum surfactant concentrations that were used toprepare single-phase antibacterial formulations during the surfactanttitrations, reflected two considerations: (1) these surfactantconcentrations optimize hydrophobe solubilization without diminishingbiocidal activity; and (2) because the turbidity increases with thesurfactant content, these compositions enable the measurement of maximumturbidity in the single-phase surfactant/microgel formulations obtainedusing each surfactant system. Here, the turbidity of dye-freesurfactant/microgel formulations was measured (without filtration atλ=488 mm) immediately after the surfactant addition, using a Cary 50spectrophotometer. Each measurement was performed three times.

Example 1

Surfactant Binding to Chitosan-Derived Microgels

The binding of anionic SDS and nonionic TX100 to the microgels wasprobed by ITC and electrophoretic light scattering. When SDS wastitrated into the microgel dispersion, an exothermic binding heat (ofapproximately 5 kJ/mol of added SDS) was detected by ITC (FIG. 1), thusshowing that the SDS was binding to the microgels. Once the SDSconcentration exceeded about 2 mmol/kg, however, the binding heatdiminished sharply to about 0.7-0.8 kJ/mol. This transition shows thesaturation of cationic binding sites. The persistence of the exothermicbinding heat beyond that point, however, shows that SDS continues tobind (through non-electrostatic interactions) even after the cationiccharges are saturated. Conversely, no binding heat was detected whenTX100 was added to the microgels, thus showing that TX100 does not bindto the chitosan-derived microgels.

This interpretation of the molecular binding events is further supportedby the ζ-potential estimates obtained by electrophoretic lightscattering. Without surfactant, the microgel ζ-potential in 10 mM NaClis about 15 mV (see FIG. 2). When SDS is added to the microgels, theζ-potential stays nearly constant, until the SDS concentration reachesthe saturation transition in the ITC data (i.e., exceeded—2 mmol/kg). Atthis point, the ζ-potential diminishes dramatically, and ultimatelybecomes negative. The microgel charge inversion that occurs after thesaturation of cationic sites confirms the continued binding of SDS tothe microgels, which occurs through hydrophobic association.

The addition of nonionic TX100 (instead of the SDS) has no effect on themicrogel ζ-potential. This combination of ITC and electrophoretic lightscattering data shows that SDS binds to the chitosan-derived microgelsand the TX100 does not.

Example 2

Colloidal Stability of Surfactant/Microgel Formulations.

To determine the surfactant compositions at which the microgels can bedispersed, the short-term colloidal stability of the surfactant/microgelformulations was investigated by titrating concentrated surfactantsolutions into dilute microgel dispersions (which initially contained8.2×10⁻² wt % HTCC). Microgel aggregation was then tracked both visuallyand by DLS. In the absence of TX-100, the microgels—whose z-averagehydrodynamic diameter was ca. 300 nm—remained dispersed until the SDSwas mixed with the cationic microgels near the saturation point ([SDS]—2mmol/kg, as shown by the ITC data and the blue diamonds in FIG. 3A).Above this point, their z-average diameters (see FIG. 3A) andpolydispersities (FIG. 9) increased sharply due to coagulation, and themicrogels precipitated. This coagulation reflects the neutralization ofthe HTCC charges on the microgel surface, which diminishes both thehydrophilicity of the microgels and the electrostatic stabilization oftheir dispersions.

When the SDS concentration was raised further, above 8 mmol/kg, themicrogels redispersed. These transitions show the interactions ofmolecular HTCC and cationically-modified hydroxyethyl cellulose withanionic surfactants—i.e., where the surfactant/polyelectrolyte complexesprecipitated when the surfactant and polymer were mixed at nearstoichiometric ratios, and were resolubilized in the limit of highsurfactant concentration. This resolubilization phenomenon is attributedto the additional binding of surfactant, which breaks up theinterpolymer surfactant/polyelectrolyte aggregates, such that thepolymer-bound surfactant aggregates are no longer shared by multiplepolymer chains or (in the case of the present example) multiplemicrogels.

The microgel hydrodynamic diameter diminished dramatically as theconcentration was increased beyond the resolubilization boundary, andmatched that obtained for SDS complexes with molecular (TPP-free) HTCC.This decrease in particle size was accompanied by a sharp reduction indispersion turbidity, thus indicating microgel dissolution andindicating that the binding of SDS can displace ionic crosslinks formedby TPP between the HTCC chains. This is further supported by comparingthe ITC data for SDS binding to HTCC/TPP microgels—where most of thebinding sites were initially occupied by TPP—with its binding tomolecular HTCC (without TPP), where all the binding sites were initiallyfree. This comparison, shown in FIG. 11, reveals that the presence ofTPP has little impact on the SDS concentration required to saturate thecationic HTCC binding sites. Thus, the electrostatic binding of SDS candisplace the TPP crosslinks within the microgels, and can lead to theirdissolution when SDS is in excess.

When the surfactant titrations were repeated using tertiarySDS/TX-100/water formulations (where the SDS:TX100 ratios were either70:30, 40:60 or 10:90), the addition of TX100 shifted the precipitationtransition to lower SDS concentrations (see FIG. 3A and FIG. 8—Table 1).

At each SDS:TX100 ratio, precipitation occurred at roughly the sameoverall surfactant concentration (roughly 1.4-2 mmol/kg, as shown inFIG. 3B and FIG. 8—Table 1). This shift of the precipitation boundary tolower SDS concentrations was particularly surprising because themicrogel ζ-potential in SDS/TX-100/water surfactant formulations wasroughly the same as that in the absence of TX100 at the same SDSconcentration (FIG. 10). This early coagulation reflects the bridging ofmicrogels with surface-bound SDS/TX100 micelles (i.e., microgel/micelleheterocoagulation), even when the cationic chitosan amine groups are inexcess. Unlike in the SDS/microgel formulations without TX-100, themicrogels did not re-disperse in SDS/TX-100 surfactant formulations athigher concentrations (up to 100 mmol/kg).

The titration experiment was repeated using nonionic TX100 as the onlysurfactant species, thereby eliminating electrostaticsurfactant/microgel binding. The microgels remained dispersed at allinvestigated surfactant concentrations ([TX-100]≦14.4 mmol/kg) and, asshown by the DLS data in FIG. 4—where the peak at 8 nm, which grows withincreasing surfactant concentration, reflects the formation of TX100micelles—appeared to have little impact on the microgel sizedistributions. This shows that surfactant-induced coagulation ofcationic microgel dispersions was avoided by the use of the nonionicsurfactant.

The short-term colloidal stability seen in this example reveals therestrictions on surfactant compositions that can be used with theHTCC/TPP microgels. In addition to the rapid, surfactant-inducedmicrogel coagulation (or disintegration), however, the long-termstability of these colloidal dispersions (which is important forcommercial use) can be undermined by other effects such as attractiveVan der Waals interactions, bridging flocculation by surface-bound TPP,or hydrolytic degradation.

Upon storage at room temperature over longer timescales (of severaldays), the microgels coagulated and precipitated at nearly all SDS andSDS/TX100 surfactant concentrations (and even without added surfactant).At low surfactant concentrations (0-2 mmol/kg), below the onset of rapidprecipitation in microgel formulations with SDS and SDS/TX-100 (see FIG.3 and Table 1 in FIG. 8), this occurred within one or two days. Thislimited colloidal stability of HTCC/TPP microgels differs greatly fromthat of non-quaternized chitosan/TPP microgels, which remain dispersedeven after several months. While not wishing to be bound by theory, itis believed that this may reflect the differences in the biopolymer/TPPbinding strength, which ITC measurements suggest to be significantlyweaker for HTCC than for chitosan (see FIG. 12). The microgelcoagulation occurs through the ionic bridging of the microgels by TPP,which occurs more rapidly at higher free TPP concentrations. The freeTPP concentration, on the other hand, scales inversely with the bindingstrength; thus, the faster coagulation of HTCC/TPP microgels reflectsthe higher concentration of bridging TPP ions in solution. This effectis also enhanced by the low ζ-potentials of HTCC/TPP microgels (˜15 mV)which, because of the weaker electrostatic repulsion, leads tomore-frequent microgel collisions.

When single-phase formulations were prepared at higher surfactantconcentrations, the colloidal stability was dramatically improved. Whenthe microgels were dispersed in 10 mmol/kg SDS (above theresolubilization phase boundary for the SDS/microgel system), themicrogels remained dispersed for more than 2 weeks at room temperature.Likewise, when the microgels were mixed with 14.4 mmol/kg TX-100, themicrogels remained dispersed for 5-6 weeks. This extended shelf lifeshows that, in addition to not causing precipitation, TX100 dramaticallyenhances the long-term colloidal stability of HTCC microgelformulations. It also indicates that, despite the lack of binding signalin the ITC data (see FIG. 1), the nonionic TX100 surfactant interactshydrophobically with the HTCC/TPP microgels.

Example 3

Antibacterial Activity of Surfactant/Microgel Formulations

The antibacterial activity of the surfactant/microgel formulations wastested using Pseudomonas aeruginosa PAO1 as a model Gram-negativebacteria. As a positive control, the bacteria were added to asurfactant-free microgel dispersion (containing 7.4×10⁻² wt % HTCC),whereupon all bacteria were rapidly killed within the first 5 min (seeFIG. 5, Group 1; where the number of viable cells, N, is normalized bytheir initial number, N₀). When SDS was added to the dispersion, suchthat the microgel cationic charges were not fully neutralized (i.e., 1mmol/kg SDS; see FIG. 5, Group 2), the microgels maintained their strongantibacterial activity and again killed all the bacteria within 5 minSimilar results were obtained when the microgels were dispersed in adilute (3 mmol/kg; see FIG. 5, Group 3) 40:60 SDS:TX100 formulation,which had a similar SDS concentration to Group 2.

The coagulation of the microgels at this SDS:TX100 surfactantconcentration (see FIG. 3) did not appear to affect their antibacterialproperties. When the SDS concentration was raised to be in excess to theHTCC amines, however (using 10 mmol/kg SDS), the antibacterial activitywas diminished, with nearly 1% of the bacteria still viable after 30 minof contact time (see FIG. 5, Group 4). This reduction in antibacterialactivity reflects the neutralization of the quaternary HTCC aminegroups—to which the antibacterial activity is typically attributed—bythe microgel-bound anionic surfactant. This neutralization is evidentfrom the ζ-potential data in FIG. 2, where the microgel charge was shownto become negative at SDS concentrations above 2.8 mmol/kg. Furthermore,because the antibacterial activity of microgels may be stronger thanthat of molecular HTCC, the reduced antibacterial activity at higher SDSconcentrations might also reflect microgel dissociation. Thus, theelectrostatic binding of anionic surfactant to the cationic microgelsdiminishes both the stability and the antibacterial activity of theformulation.

Conversely, when an excess (14.4 mmol/kg) of the non-binding TX100 wasadded to the microgels, the strong antibacterial properties werepreserved (see FIG. 5, Group 5), and all of the bacteria were killedwithin 5 min of contact time. This shows that, by avoiding electrostaticbinding, antibacterial activity of chitosan-based microgels can bepreserved even at high surfactant concentrations.

To ensure that the surfactants used were not biocidal on their own, thecells were exposed to microgel-free SDS, TX100 and SDS/TX100 surfactantsolutions (see FIG. 5, Groups 6-8) and water (see FIG. 5, Group 9) asnegative controls. These measurements confirmed that, without themicrogels, the surfactants (see FIG. 5, Groups 6-8) had no short-termeffect on bacterial viability, and that the rapid antibacterial activityof the surfactant/microgel formulations stems from the microgels and notthe surfactants.

Likewise, when the bacteria were exposed to water (without surfactant ormicrogels; see FIG. 5, Group 9), the viable cell counts remainedconstant over the 30 min experiment. These results show that the strongantibacterial properties of chitosan-based microgels are mediated bytheir cationic amine groups. Accordingly, in formulations containingnonionic and anionic surfactants, desirable antibacterial activity isachieved using either nonionic surfactants, or anionic surfactants atlow concentrations (i.e., where the microgel amine groups are notneutralized).

Example 4

Hydrophobe Solubilization Properties of Surfactant/Microgel Formulations

In addition to their colloidal stability and antibacterial properties,it is desirable that successful cleaning and personal care productformulations be able to solubilize hydrophobic compounds. To this end,hydrophobe solubilization properties of microgel formulations with SDS,SDS/TX100 and TX100 solutions were measured. The surfactantconcentrations in dispersions prepared using the SDS and SDS/TX100surfactant systems were selected to be near the onset of rapidprecipitation (see FIG. 3B), such that the surfactant content was nearthe maximum that can be used without undermining the phase stability orantibacterial activity of the dispersion. Similarly, dispersionsprepared using only TX100 —where no rapid precipitation or antibacterialactivity reduction occurred—were prepared at the maximum TX100concentration used in the colloidal stability and antibacterial activitytests (i.e., 14.4 mmol/kg TX100).

To test the hydrophobe solubilization properties of thesurfactant/microgel formulations, the dispersions were mixed withhydrophobic blue (guaiazulene) dye. When the dye was mixed with thesurfactant/microgel formulations, a fraction of the dye dissolved insamples containing TX100 or SDS/TX100 formulations. This was shown bythe blue color of the surfactant/microgel formulation (see FIG. 6A,Samples ii-v), and confirmed by UV-Vis spectroscopy (FIG. 6B). No dyedissolution, however, occurred in dispersions containing SDS withoutTX100 (FIG. 6A, Sample i, and FIG. 6A).

Microgel dispersions prepared using 70:30 and 40:60 SDS:T′X-100 ratiosalso generated finely-dispersed dye particles, which were notsolubilized at the molecular level. Most of these particles (along withthe microgels) were removed by filtration through a 0.2 μm filter priorto spectroscopic analysis. The dye particles at the 40:60 SDS:TX100ratio, however, were dispersed more-finely than those at 70:30SDS:TX-100. Consequently, the filtration of 70:30 SDS:TX100 samplesyielded clear solutions, while the filtered 40:60 SDS:TX100 samplesremained opaque. This indicated that nearly all undissolved particleswere removed from the 70:30 SDS:TX100 sample, while in the 40:60SDS:TX100 samples some of the dye particles remained dispersed. Thepresence of finely-dispersed dye particles in the 40:60 SDS:TX-100samples made the measured solubilized dye concentration (in FIG. 6B)appear higher than it really was (and higher than it was in otherSDS-bearing samples). Because of this artifact, the true dye solubilityin the 40:60 SDS:TX-1001 microgel formulation is lower than it appearsin the UV-Vis measurements. The microgels dispersed in 14.4 mmol/kgTX-100, however, had by far the best solubilization properties (wherethe solubilized dye concentration was almost seven times higher thanthat measured for the 40:60 SDS:TX100 samples).

The lack of dissolved dye in dispersions containing only SDS reflectedthe fact that almost all of the SDS was taken up by the microgels, andwas therefore unavailable to interact with the dye. The impact of SDSuptake on dye solubilization was confirmed by mixing the dye withmicrogel-free 1.75 mmol/kg SDS (i.e., the SDS concentration in FIG. 6A,Sample i), which yielded a dark blue mixture with finely-dispersed dyeparticles (see FIG. 13).

Conversely, when SDS/TX100 formulations were used, a substantial portionof the surfactant remained outside the microgels, and enabled dyesolubilization. The stable dye particle dispersions that form at 70:30and 40:60 SDS:TX100 ratios reflects the electrostatic repulsion thatexists between dye particles coated with anionic surfactant, and showsthat the surfactant/microgel formulations are able to remove hydrophobicsoils without molecular solubilization. However, because the SDS-freemicrogel/TX100 formulation can be prepared at higher surfactantconcentrations (without compromising colloidal stability orantibacterial activity), the nonionic surfactant-based formulations(e.g., FIG. 6A, Sample v) provide the best solubilization properties.

Example 5

Effects of Surfactant on Mixture Turbidity

Besides being functional, it is desirable that consumer productformulations be aesthetically appealing. For products sold in clearpackaging, clarity is a key optical property.

The effect of surfactant on the turbidity of the chitosan-based microgeldispersions was characterized at the same surfactant concentrations aswere used in the hydrophobe solubilization tests (see FIG. 7).

The dispersion clarity depended strongly on the SDS content. When onlySDS was added to the microgels (without TX100), the turbidity increaseddramatically, despite the nearly-constant hydrodynamic diameter of themicrogels (see FIG. 3). This reflects a greater refractive indexcontrast between the microgel and solvent phases, which stems from theuptake of organic SDS molecules into the aqueous microgel particles.

When SDS/TX100 surfactant formulations were used, the turbidity increasebecame progressively less-pronounced with decreasing SDS:TX100 ratios,showing a smaller extent of surfactant uptake by the microgels.Likewise, when SDS-free TX100 was used, the turbidity remained virtuallyunchanged by the addition of surfactant. This result shows that theclarity of the nonionic surfactant/microgel formulations is notdiminished by the uptake of surfactants into the microgels (like it isin the case of anionic surfactants), and that microgel dispersions withnonionic surfactants exhibit superior optical properties to their ionicsurfactant-containing counterparts.

Thus, these examples show that the chitosan-derived microgels are highlyantibacterial, and are useful alternatives to cationic surfactants andother low-molecular-weight antibacterial agents. In addition, the mixingof chitosan-derived microgels with a nonionic surfactant (TX100)preserves the chitosan-derived microgel's antibacterial activity and lowoptical density, does not lead to rapid, surfactant-inducedprecipitation (which limits the range of surfactant concentrations thatcan be used), and enhances long-term colloidal stability.

Furthermore, because the addition of nonionic surfactant does notundermine the biocidal activity and colloidal stability of themicrogels, the nonionic surfactant/microgel formulations can be preparedat high surfactant concentrations, which provide superior hydrophobesolubilization.

Conversely, chitosan-based microgel formulations with anionic (SDS) andanionic/nonionic (SDS/TX100) surfactant formulations undergo rapid,surfactant-induced precipitation. In the case of mixed anionic/nonionicsurfactant systems this limits the single-phase formulation compositionsto very low surfactant concentrations, which lead to poor hydrophobesolubilization properties.

When microgels are mixed with only anionic surfactant, however, theformulations remain in a single phase both in the limits of low and highsurfactant concentrations (with surfactant-induced precipitationoccurring at intermediate concentrations). As in the case of theanionic/nonionic surfactant formulations, formulations at low anionicsurfactant concentration exhibit poor hydrophobe solubilizationproperties (which are worse than those in anionic/nonionic surfactantformulations).

Conversely, at higher anionic surfactant concentrations (above the CMC,where single-phase mixtures again form), the binding of surfactant tothe microgels weakens their biocidal activity and ultimately leads tomicrogel dissolution. Furthermore, the uptake of anionic surfactant bythe microgels makes the microgels more opaque, thereby potentiallymaking the anionic surfactant/microgel formulations less-suitable forclear packaging.

Also, HTCC/TPP microgels mixed with anionic surfactant (or withoutsurfactant) exhibit poor long-term colloidal stability, and coagulatewithin days. These results show that stable formulations that combineantibacterial activity with good solubilization and optical properties,surfactant/chitosan-based microgel formulations are achieved whennonionic surfactant systems are used in the formulations.

Example 6

Evolution in Microgel Polydispersity

FIG. 9 shows the evolution in the microgel polydispersity index (PDI)during the titration of surfactant (either SDS or SDS/TX100 at 70:30,40:60 and 10:90 SDS:TX100 ratios). At low surfactant concentrations, theaddition of surfactant gradually diminishes the PDI. At the onset ofprecipitation, however (where the microgels begin to rapidly coalesce),the PDI-values increase sharply. The sharp increase in PDI-valuesreflects the formation of polydisperse microgel flocs.

Example 7

Evolution in ζ-Potential

FIG. 10 shows the evolution in the microgel ζ-potential during thetitration of surfactant (either SDS or SDS/TX100 in 70:30, 40:60 and10:90 SDS:TX100 ratios), plotted as a function of SDS concentration. Theoverlap between the data points collected at different SDS:TX100 ratiosreveals that (despite the effect of TX100 on microgel coagulation) themicrogel ζ-potential is unaffected by TX-100.

Example 8

Critical Micelle Concentrations of SDS/TX-100 Formulations

To confirm that coagulation in SDS/TX-100/microgel formulations occurredat surfactant concentrations above the surfactant critical micelleconcentration (CMC), the CMCs of SDS/TX100 formulations in 10 mM NaClwere measured by isothermal titration calorimetry (ITC). The results ofthese measurements are presented in FIG. 11, which shows that themicrogels coagulate (and precipitate) at surfactant concentrations abovethe CMC.

Example 9

ITC Analysis of TPP Binding to HTCC

The strength of chitosan/ionic crosslinker binding can be shown fromisothermal titration calorimetry (ITC) data. FIG. 12 compares the ITCdata for TPP binding to chitosan (red circles) and HTCC (which was usedto prepare the microgels; black squares). The data in these plots areroughly proportional to the first derivative of the binding isothermwhen plotted as a function of total binding ligand (in this case TPP)concentration. Consequently, the abruptness of the saturation transition(where the exothermic binding signal diminishes to approximately 0kJ/mol) reflects the binding strength of each polymer/crosslinkersystem. Comparing these transitions for the two data sets—where thesaturation transition for the chitosan/TPP system is much more abruptthan that for the HTCC/TPP system—shows that the binding of TPP to HTCCis much weaker than its binding to chitosan.

Example 10

Dye Solubilization in Microgel-Free SD

FIG. 13 shows a photograph of hydrophobic (guaiazulene) blue dyeequilibrated for 24 h with 1.75 mmol/kg SDS solution. The uniform bluecolor in this formulation is in stark contrast with the clearsupernatant that forms in 1.75 mmol/kg SDS solution (FIG. 6A, Sample i),and shows that the inability of this SDS/microgel formulation tosolubilize the dye stems from the uptake of surfactant into themicrogel.

Example 11

Polyelectrolyte Complexation

The antibacterial quaternized chitosan derivative,N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC), wasprepared as described above. HTCC, sodium alginate, Triton X-100(TX100), and DI water were used in this example to prepare microgels bypolyelectrolyte complexation, and then to prepare surfactant/microgelformulations with the microgels formed through polyelectrolytecomplexation.

To prepare a microgel mixture, 3.5 mL of 0.1 wt % alginate solution wasadded dropwise at an approximate rate of 200 μL/min to 10 mL of 0.01 wt% HTCC solution. The addition was conducted with a pipette, and thereceiving solution was stirred with a magnetic stir bar at 800 rpm in a20 mL scintillation vial. After 10 min of equilibration, before thesurfactant was added, the microgel exhibited a positive zeta potentialof about 30 mV. Following equilibration, 1.3 mL of 0.1 M TX100 solutionwas added to the microgel mixture at an approximate rate of 1 mL/min Theresulting mixture was equilibrated for 10 min

The above procedure was repeated with varying alginate:HTCC ratios andvarying concentrations of HTCC solution. FIGS. 14A-14C show the sizedistributions (z-average diameters and polydispersity indices) and zetapotentials of the HTCC/alginate particles prepared at varyingalginate:HTCC ratios. FIGS. 15A-15D show the size distributions, zetapotentials, and derived count rate of the HTCC/alginate particlesprepared with these varying concentrations of HTCC. Concentrations of0.01 wt %, 0.02 wt %, 0.03 wt %, 0.04 wt %, and 0.05 wt % HTCC wereused.

The antibacterial activity of mixtures of surfactant and HTCC/alginate,prepared as described above, was tested using Pseudomonas aeruginosaPAO1 as model Gram-negative bacteria. These tests showed that themixtures of surfactant and HTCC/alginate particles have antibacterialactivity. (FIGS. 16A-16B.) As seen in FIGS. 16A-1B, exposure to thesurfactant/microgel mixture prepared from polyelectrolyte complexationsignificantly reduced the percent of viable bacterial cells after a veryshort time frame.

Example 12

Exemplary Uses

Non-limiting uses include medical, dental, personal care and industrialuses. Non-limiting specific examples include: textiles, food andbeverages; biopharmaceuticals, medical implants, wound dressings,dentistry implants and implements, packaging materials, article,compound, composition, film, yarn, fabric, mesh, screen material and thelike. Other uses include use as cleaning agents, antibacterial agents,antiseptic agents, and the like.

It is to be understood that the formulations described herein may be theform of a tablet, powder, gel, capsule, liquid, coating, film, foam,sponge, woven material, non-woven material, knitted material, textiles,papers, porous material or solid material.

Also, within the contemplated scope of the invention herein are articlescomprising a porous or solid material at least partially coated with theformulation; and/or at least partially comprised of the formulation.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to the particular embodimentdisclosed herein contemplated for carrying out this invention, but thatthe invention will include all embodiments falling within the scope ofthe claims.

What is claimed is:
 1. An antibacterial formulation comprising: aquaternized polymeric microgel comprising a cationic polymer complexedwith an anionic polymer; and at least one surfactant capable ofproviding colloidal stability to the formulation.
 2. The antibacterialformulation of claim 1, wherein the cationic polymer comprisesN-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC). 3.The antibacterial formulation of claim 1, wherein the surfactant is anonionic surfactant.
 4. The antibacterial formulation of claim 3,wherein the nonionic surfactant comprises TX-100.
 5. The antibacterialformulation of claim 1, wherein the surfactant is an anionic surfactantselected from the group consisting of: sodium dodecyl sulfate (SDS),sodium laurate, sodium stearate, dioctyl sodium sulfosuccinate,amphoteric sodium N-lauroyl sarcosinate, and mixtures thereof.
 6. Theantibacterial formulation of claim 1, wherein the surfactant comprises amixture of at least one anionic surfactant and at least one nonionicsurfactant.
 7. The antibacterial formulation of claim 1, wherein theformulation is essentially free of cationic surfactants.
 8. Theantibacterial formulation of claim 1, wherein the cationic polymer ispresent at a concentration ranging from about 0.001% by weight to about10% by weight, and the anionic polymer is present at a concentration upto about 1,500 mM.
 9. The antibacterial formulation of claim 1, whereinthe anionic polymer comprises alginate.
 10. The antibacterialformulation of claim 1, wherein the anionic polymer comprises celluloseor a cellulose derivative.
 11. The antibacterial formulation of claim 1,wherein the anionic polymer comprises carboxymethyl cellulose.
 12. Theantibacterial formulation of claim 1, wherein the quaternized polymericmicrogel is present at a concentration ranging from about 0.01% byweight to about 0.5% by weight.
 13. The antibacterial formulation ofclaim 1, wherein the quaternized polymeric microgel comprises particleshaving an average diameter ranging from about 120 nm to about 180 nm.14. A microgel mixture comprising a polyelectrolyte complex between aquaternized chitosan polymer and an anionic polymer, wherein the anionicpolymer is selected from the group consisting of alginate, cellulose,and derivatives thereof.
 15. An antibacterial formulation comprising themicrogel mixture of claim
 14. 16. A method of making an antibacterialformulation, the method comprising: forming a polyelectrolyte complexbetween a cationic polymer and an anionic polymer; and dispersing thepolyelectrolyte complex in a surfactant to make an antibacterialformulation.
 17. The method of claim 16, wherein the surfactant is anonionic surfactant.
 18. The method of claim 17, wherein the nonionicsurfactant comprises t-octylphenoxypolyethoxyethanol (TX100).
 19. Themethod of claim 16, wherein the surfactant is an anionic surfactantselected from the group consisting of: sodium dodecyl sulfate (SDS),sodium laurate, sodium stearate, dioctyl sodium sulfosuccinate,amphoteric sodium N-lauroyl sarcosinate, and mixtures thereof.
 20. Themethod of claim 16, wherein the surfactant comprises a mixture of atleast one anionic surfactant and at least one nonionic surfactant. 21.The method of claim 16, wherein the cationic polymer comprises aquaternized chitosan polymer, and the anionic polymer comprisesalginate.
 22. The method of claim 16, wherein the cationic polymercomprises a quaternized chitosan polymer, and the anionic polymercomprises carboxymethyl cellulose.
 23. The method of claim 16, whereinthe polyelectrolyte complex is allowed to equilibrate for a period oftime before being dispersed in the surfactant.
 24. The method of claim16, wherein the surfactant comprises TX100 and the polyelectrolytecomplex comprises particles of HTCC complexed with alginate.
 25. Themethod of claim 16, wherein the surfactant comprises TX100 and thepolyelectrolyte complex comprises particles of HTCC complexed withcarboxymethyl cellulose.
 26. The product of the method of claim 16.